Limits...
Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways.

Euba B, Moleres J, Viadas C, Ruiz de los Mozos I, Valle J, Bengoechea JA, Garmendia J - PLoS ONE (2015)

Bottom Line: Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20.We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant.Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain.

ABSTRACT
Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Show MeSH

Related in: MedlinePlus

Infection of respiratory epithelial cells by NTHi375 mutant strains lacking the ompP5 and hap genes.Bacterial adhesion is shown in the left and invasion in the right panels, in all sections of this figure. (A) NTHi375 interplay with upper and lower human epithelial airways. NTHi375 adhesion to- and invasion of nasal RPMI 2650, pharynx Detroit 562 and bronchial H-292 epithelia and type II pneumocytes A549. Effect of ompP5 and hap gene disruption on NTHi interplay with RPMI 2650 nasal (B), Detroit 562 pharynx (C), NCI H-292 bronchial (D), and A549 type II alveolar (E) epithelial cells. NTHi375, ΔompP5, Δhap and ΔompP5Δhap strains were used to assess bacterial adhesion and invasion. (F) Effect of ompP5 gene disruption on Hi RdKW20 interplay with NCI H-292 bronchial and A549 type II alveolar epithelial cells. Hi RdKW20 and ΔompP5 mutant strains were used to assess bacterial adhesion and invasion. Experiments were performed in triplicate in at least three independent occasions (n≥9).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403991&req=5

pone.0123154.g002: Infection of respiratory epithelial cells by NTHi375 mutant strains lacking the ompP5 and hap genes.Bacterial adhesion is shown in the left and invasion in the right panels, in all sections of this figure. (A) NTHi375 interplay with upper and lower human epithelial airways. NTHi375 adhesion to- and invasion of nasal RPMI 2650, pharynx Detroit 562 and bronchial H-292 epithelia and type II pneumocytes A549. Effect of ompP5 and hap gene disruption on NTHi interplay with RPMI 2650 nasal (B), Detroit 562 pharynx (C), NCI H-292 bronchial (D), and A549 type II alveolar (E) epithelial cells. NTHi375, ΔompP5, Δhap and ΔompP5Δhap strains were used to assess bacterial adhesion and invasion. (F) Effect of ompP5 gene disruption on Hi RdKW20 interplay with NCI H-292 bronchial and A549 type II alveolar epithelial cells. Hi RdKW20 and ΔompP5 mutant strains were used to assess bacterial adhesion and invasion. Experiments were performed in triplicate in at least three independent occasions (n≥9).

Mentions: To assess the role of P5 and Hap in NTHi interaction with the human airways epithelia, we used nasal RPMI 2650, pharynx Detroit 562, bronchial NCI H-292 and A549 type II pneumocytes cultured epithelial cells. First, we established infection levels for NTHi375 wild-type strain in the four cell types. NTHi375 adhered to the four cell types with variable numbers. Adhesion to nasal cells was significantly lower than that observed for the other cell types tested (mean numbers for RMPI 2650 were lower than those obtained for Detroit 562 and H-292 (p<0.0001), and for A549 (p<0.05) cells); adhesion to Detroit 562 was significantly lower than to NCI H-292 (p<0.0001), but higher than to A549 (p<0.0001) cells; adhesion to NCI H-292 was higher than that observed for the other cell types tested (p<0.0001) (Fig 2A). In terms of NTHi375 internalization into the four cell types, the highest invasion was found for NCI H-292 (p<0.0001) cells, followed by pharynx, nasal and alveolar cells, respectively. Thus, NTHi375 invasion of RMPI 2650 cells was lower than that obtained for Detroit 562 (p<0.05) and H-292 (p<0.0001) cells, and higher than that shown by A549 (p<0.005) cells; invasion of Detroit 562 was lower than that obtained for H-292 (p<0.0001), and higher than the one measured for A549 (p<0.0005) cells (Fig 2A).


Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways.

Euba B, Moleres J, Viadas C, Ruiz de los Mozos I, Valle J, Bengoechea JA, Garmendia J - PLoS ONE (2015)

Infection of respiratory epithelial cells by NTHi375 mutant strains lacking the ompP5 and hap genes.Bacterial adhesion is shown in the left and invasion in the right panels, in all sections of this figure. (A) NTHi375 interplay with upper and lower human epithelial airways. NTHi375 adhesion to- and invasion of nasal RPMI 2650, pharynx Detroit 562 and bronchial H-292 epithelia and type II pneumocytes A549. Effect of ompP5 and hap gene disruption on NTHi interplay with RPMI 2650 nasal (B), Detroit 562 pharynx (C), NCI H-292 bronchial (D), and A549 type II alveolar (E) epithelial cells. NTHi375, ΔompP5, Δhap and ΔompP5Δhap strains were used to assess bacterial adhesion and invasion. (F) Effect of ompP5 gene disruption on Hi RdKW20 interplay with NCI H-292 bronchial and A549 type II alveolar epithelial cells. Hi RdKW20 and ΔompP5 mutant strains were used to assess bacterial adhesion and invasion. Experiments were performed in triplicate in at least three independent occasions (n≥9).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403991&req=5

pone.0123154.g002: Infection of respiratory epithelial cells by NTHi375 mutant strains lacking the ompP5 and hap genes.Bacterial adhesion is shown in the left and invasion in the right panels, in all sections of this figure. (A) NTHi375 interplay with upper and lower human epithelial airways. NTHi375 adhesion to- and invasion of nasal RPMI 2650, pharynx Detroit 562 and bronchial H-292 epithelia and type II pneumocytes A549. Effect of ompP5 and hap gene disruption on NTHi interplay with RPMI 2650 nasal (B), Detroit 562 pharynx (C), NCI H-292 bronchial (D), and A549 type II alveolar (E) epithelial cells. NTHi375, ΔompP5, Δhap and ΔompP5Δhap strains were used to assess bacterial adhesion and invasion. (F) Effect of ompP5 gene disruption on Hi RdKW20 interplay with NCI H-292 bronchial and A549 type II alveolar epithelial cells. Hi RdKW20 and ΔompP5 mutant strains were used to assess bacterial adhesion and invasion. Experiments were performed in triplicate in at least three independent occasions (n≥9).
Mentions: To assess the role of P5 and Hap in NTHi interaction with the human airways epithelia, we used nasal RPMI 2650, pharynx Detroit 562, bronchial NCI H-292 and A549 type II pneumocytes cultured epithelial cells. First, we established infection levels for NTHi375 wild-type strain in the four cell types. NTHi375 adhered to the four cell types with variable numbers. Adhesion to nasal cells was significantly lower than that observed for the other cell types tested (mean numbers for RMPI 2650 were lower than those obtained for Detroit 562 and H-292 (p<0.0001), and for A549 (p<0.05) cells); adhesion to Detroit 562 was significantly lower than to NCI H-292 (p<0.0001), but higher than to A549 (p<0.0001) cells; adhesion to NCI H-292 was higher than that observed for the other cell types tested (p<0.0001) (Fig 2A). In terms of NTHi375 internalization into the four cell types, the highest invasion was found for NCI H-292 (p<0.0001) cells, followed by pharynx, nasal and alveolar cells, respectively. Thus, NTHi375 invasion of RMPI 2650 cells was lower than that obtained for Detroit 562 (p<0.05) and H-292 (p<0.0001) cells, and higher than that shown by A549 (p<0.005) cells; invasion of Detroit 562 was lower than that obtained for H-292 (p<0.0001), and higher than the one measured for A549 (p<0.0005) cells (Fig 2A).

Bottom Line: Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20.We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant.Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain.

ABSTRACT
Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Show MeSH
Related in: MedlinePlus